For the treatment of acute and chronic liver failure, there is a critical need for both temporary and more permanent modes of liver support. Hepatocyte-based liver support systems offer a viable alternative for both of these modalities but such a program relies heavily on a consistent, abundant, and readily available supply of hepatocytes when the need for liver support arises. Thus, proper hepatocyte preservation techniques are critical if such an extracorporeal device is ever to become feasible. The best and most promising method currently available for preservation of living systems is cryopreservation. Unfortunately, there are no reproducible methodologies for cryopreservation of isolated hepatocytes which result in long-term viability and differentiated function after a freeze-thaw cycle. Our preliminary investigations with rat hepatocytes cultured between two collagen layers (sandwich culture) have produced promising results. However, further efforts must be undertaken to understand and optimize the cryopreservation process for isolated hepatocytes, especially for human hepatocytes. In order to optimize the cryopreservation of hepatocytes, the fundamental physicochemical processes must be better understood. Research in cryobiology has shown that the response of cells to the thermodynamic changes of state imposed by the freezing protocol are unique to a cell type and are a consequence of the competing rate processes of heat transfer and water transport. These rate processes must be determined in the hepatocyte, especially human hepatocyte, and cannot be extrapolated from other cell types. The specific aims of the proposed study are to: (1) determine the fundamental biophysical parameters which govern the freezing behavior of isolated rat and human hepatocytes, and use these parameters in a thermodynamic model to predict optimal cooling protocols; (2) further improve the cryopreservation protocols developed in Specific Aim #1 to decrease the sensitivity of hepatocytes to freezing by optimizing a number of critical pre-freeze and post-thaw conditions; (3) characterize the long-term effects of cryopreservation on frozen-thawed isolated rat and human hepatocytes with the major emphasis on differentiated functions; (4) develop methodologies for cryopreservation of human hepatocytes cultured in the stable differentiated sandwich culture configuration; and (5) test the efficacy of cryopreservation protocols using animal models. The principles developed in this study will provide a rationale basis for the design of reversible cryopreservation protocols for rat and human hepatocytes, and will facilitate the clinics applications of hepatocyte- based artificial liver support systems.